Bottom Line:
Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX).Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis.It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway.

ABSTRACTNon-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG2000-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.

Mentions:
GE11 was conjugated to DSPE-PEG2000-Mal by an addition reaction and the structure of the product was verified by 1H-NMR and Fourier transform infrared spectroscopy. As shown in Figure 1A, the peaks at 0.81 ppm and 1.23 ppm were attributed to methyl and methylene protons in DSPE, while the peaks at 3.53 ppm and 6.5–9.3 ppm were attributed to PEG and GE11, respectively. In the Fourier transform infrared spectrum (Figure 1B), the peak at 1,105 cm−1 was the stretching vibration of C–O–C in the PEG segment and the peak at 1,679 cm−1 was the stretching vibration of C(=O)–N in GE11. These results suggested that GE11 was successfully conjugated to DSPE-PEG2000-Mal.

Mentions:
GE11 was conjugated to DSPE-PEG2000-Mal by an addition reaction and the structure of the product was verified by 1H-NMR and Fourier transform infrared spectroscopy. As shown in Figure 1A, the peaks at 0.81 ppm and 1.23 ppm were attributed to methyl and methylene protons in DSPE, while the peaks at 3.53 ppm and 6.5–9.3 ppm were attributed to PEG and GE11, respectively. In the Fourier transform infrared spectrum (Figure 1B), the peak at 1,105 cm−1 was the stretching vibration of C–O–C in the PEG segment and the peak at 1,679 cm−1 was the stretching vibration of C(=O)–N in GE11. These results suggested that GE11 was successfully conjugated to DSPE-PEG2000-Mal.

Bottom Line:
Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX).Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis.It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway.

ABSTRACTNon-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG2000-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.